Because the gastrointestinal mucosa is a critical early site of viral replication and CD4 T-cell depletion, as well as the largest lymphoid organ in the body, assessment of the adaptive immune mechanisms functioning in this tissue may provide important new insights relevant to our understanding of the host-pathogen relationship. Our studies during the prior funding cycle indicated that a robust, often polyfunctional CD8 T-cell response is mounted in gut mucosa during chronic HIV infection. In the next funding cycle, we will build upon prior studies to test three complementary hypotheses concerning the role of mucosal immunity in HIV pathogenesis. Our studies will focus on (1) Polyfunctional mucosal T-cells as an immune correlate of non-progression; (2) The effects of antiretroviral therapy (ART) on CD8 T-cell phenotype and function in the gut; and (3) The role of mucosal regulatory T-cells in shaping polyfunctional adaptive immune responses. These studies will be conducted on paired blood and mucosal samples from HIV+ subjects on and off ART, including a well-characterized group of HIV controllers, and healthy controls. In Specific Aim 1, we will test the hypothesis that polyfunctional, HIV-specific CD8 T-cells in mucosal tissues are an immune correlate of non-progression. For the purposes of this study, we define polyfunctional T cells as those capable of secreting multiple cytokines/chemokines and releasing cytolytic granules upon in vitro stimulation. Our hypothesis predicts that mucosal T-cell responses in individuals characterized as Elite Controllers (EC, viral load <75) and Viremic Controllers (VC, viral load 75-2,000) are stronger, more functionally complex, and broader than in Non-Controllers (NC, viral load >10,000). For Specific Aim 2, we will focus on Non-Controllers who are beginning ART. We will test the hypotheses that (a) the gut microenvironment drives virus-specific memory CD8+ T-cells to immune senescence due to high antigen load, CD4 T-cell depletion, and local expression of ligands for PD- 1; and (b) ART will lead to only partial CD4 restoration and reversal of immune senescence in the gut. Finally, for Specific Aim 3, we will test the hypothesis that regulatory T-cells are present in mucosal tissues of HIV-infected individuals, and that these cells act locally within mucosal tissues to suppress HIV-specific effector functions. Taken together, these studies should greatly advance our understanding of the HIV-host relationship in mucosal tissues during chronic infection.